NMR spectroscopy suffers from an extremely serious disadvantage: very low sensitivity. The goal of this project is to develop novel approaches to enhance the sensitivity of liquid state nuclear magnetic resonance (NMR) and optimize the detection limits of biomolecules containing aromatic amino acids. This research will take advantage of method developments in heteronuclear correlation photo-chemically-induced dynamic nuclear polarization, also known as photo-CIDNP, and it will employ NMR setups in conjunction with an Argon Ion laser able to photo-excite flavin-containing NMR samples at 488 nm. The following three specific research aims will be pursued. Specific Aim #1: Development of novel 13C-based photo-CIDNP pulse sequences involving 13C-1H heteronuclear correlation. Several novel 2D pulse sequences will be developed and tested on both free aromatic amino acids (including Trp, which exhibits a strong 1H photo-CIDNP) and aromatic amino acids belonging to the model protein apoHmpH. The effect of laser power (up to 25W), laser duration, temperature, and viscosity will also be systematically explored. We perceive this aim as extremely exciting, in light of our recent results on 15N1H heteronuclear correlation and preliminary ab initio calculations, which strongly support the feasibility of this approach. Specific Aim #2: Ab initio electron density calculations on model substituted indoles and other aromatic compounds resembling the side chain of naturally occurring amino acids and amino acid analogs. These calculations will be based on restricted Hartree-Fock and Density Functional approaches. The computations will be carried out with the Gaussian software package, available in the UW-Madison Department of Chemistry. The goal of this aim is to evaluate the hyperfine couplings of radical cations involved in photo-CIDNP, for the relevant nuclei of interest. The results of these studies will inform the method developments in Specific Aim #1. Specific Aim #3: Application of the methods developed in Specific Aim #1 to study the binding of polypeptides rich in aromatic amino acids to the bacterial trigger factor (TF) molecular chaperone at low concentrations. In addition to the TF from E. coli, an aromatic-rich polypeptide derived from the Hmp protein (comprising its residues 116-134) will be utilized for these studies. The proposed studies are aiming at pushing the sensitivity boundaries of liquid state NMR under mild, physiologically relevant conditions suitable for biological applications in dilute solutions. PUBLIC HEALTH RELEVANCE: This project targets novel approaches to enhance the sensitivity of liquid state nuclear magnetic resonance (NMR) and optimize the detection limits of biomolecules containing aromatic amino acids by photo-chemically- induced dynamic nuclear polarization (photo-CIDNP). Given the applicability of this approach to both aromatic amino acids within proteins and aromatic-ring-containing antibiotics, this research has direct implications for the sensitive detection of antibiotic binding to their biomolecular targets, one of the aims of NIAID. The proposed applications to the bacterial chaperone trigger factor (TF) are relevant to the bacterial translation machinery, a frequent target for antibiotic development.